In Vivo Method for Generating Diversity in a Protein Scaffold

ABSTRACT

A transgenic non-human animal is provided. In certain embodiments, the animal comprises a genome comprising an immunoglobulin heavy chain locus comprising: a) a transcribed gene encoding a fusion protein comprising, from N-terminus to C-terminus: i. a scaffold comprising a first binding domain; and ii. a heavy chain constant region operably linked to the scaffold; wherein the scaffold is capable of specifically binding to a target in the absence of additional polypeptides; and b) a plurality of pseudogenes that are operably linked to the transcribed gene and that donate, by gene conversion, nucleotide sequence to the part of the transcribed gene that encodes the binding domain.

BACKGROUND

Many types of proteins have the capacity to serve as a scaffold for thecreation of new binding proteins that can be used as a therapeutic ordiagnostic. Such scaffolds generally contain a relatively invariant“framework” region that provides structure to the scaffold, and othermore substitution-tolerant regions that make contact with and providefor specific binding to a target. The amino acid sequence of the contactregions are typically different for each target. The contact regions maybe solvent exposed, and can be adjacent to each other or on oppositesides of the scaffold protein, depending on the nature of the scaffold.Due to the wide range of structures, there is considerable opportunityto develop custom molecules with commercial application. Indeed, thereare engineered scaffolds currently in clinical development.

Current scaffold methodologies generally lack an in vivo process bywhich both genetic diversification and clonal selection can occur.

SUMMARY

A transgenic non-human animal is provided. In certain embodiments, theanimal comprises a genome comprising an immunoglobulin heavy chain locuscomprising: a) a transcribed gene encoding a fusion protein comprising,from N-terminus to C-terminus: i. a scaffold comprising a first bindingdomain; and ii. a heavy chain constant region operably linked to thescaffold; wherein the scaffold is capable of specifically binding to atarget in the absence of additional polypeptides; and b) a plurality ofpseudogenes that are operably linked to the transcribed gene and thatdonate, by gene conversion, nucleotide sequence to the part of thetranscribed gene that encodes the binding domain.

In some embodiments, the animal may additionally comprise animmunoglobulin light chain locus that encodes a light chain constantregion but not a light chain variable domain, where the fusion proteinencoded by the heavy chain locus and the light chain constant regionencoded by the light chain locus, when expressed, link together via adisulfide bond in the same was a classical antibody.

In other embodiments, the animal may additionally comprise animmunoglobulin light chain locus comprising: a) a second transcribedgene encoding a second fusion protein comprising, from N-terminus toC-terminus: i. a second scaffold comprising a second binding domain; andii. a light chain constant region operably linked to the scaffold;wherein the scaffold is capable of specifically binding to a target inthe absence of additional polypeptides; and b) a plurality ofpseudogenes that are operably linked to the second transcribed gene andthat donate, by gene conversion, nucleotide sequence to the part of thesecond transcribed gene that encodes the second binding domain. In theseembodiments, the first and second binding domains may have differentbinding specificities.

Method for making fusion proteins that employ the subject animal, aswell as fusion proteins made by the same are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates an example of a subject immunoglobulinheavy chain locus.

FIG. 2 schematically illustrates several types of binding proteins

FIGS. 3A and 3B schematically illustrate a strategy for constructing anexample of a subject immunoglobulin heavy chain locus.

FIG. 4 shows an electroblot probed with anti-chicken IgY antibody

DEFINITIONS

The terms “determining”, “measuring”, “evaluating”, “assessing” and“assaying” are used interchangeably herein to refer to any form ofmeasurement, and include determining if an element is present or not.These terms include both quantitative and/or qualitative determinations.Assessing may be relative or absolute. “Determining the presence of”includes determining the amount of something present, as well asdetermining whether it is present or absent.

The term “gene” refers to a nucleic acid sequence comprised of apromoter region, a coding sequence, and a 3′UTR.

The terms “protein” and “polypeptide” are used interchangeably herein.

A “leader sequence” is a sequence of amino acids present at theN-terminal portion of a protein which facilitates the secretion of themature form of the protein from the cell. The definition of a signalsequence is a functional one. The mature form of the extracellularprotein lacks the signal sequence, which is cleaved off during thesecretion process.

The term “nucleic acid” encompasses DNA, RNA, single stranded or doublestranded and chemical modifications thereof. The terms “nucleic acid”and “polynucleotide” are used interchangeably herein.

A “non-human” animal refers to any animal of a species that is nothuman.

The term “progeny” or “off-spring” refers to any and all futuregenerations derived and descending from a particular animal. Thus,progeny of any successive generation are included herein such that theprogeny, the F1, F2, F3, generations and so on are included in thisdefinition.

The phrase “transgenic animal” refers to an animal comprising cellscontaining foreign nucleic acid (i.e., recombinant nucleic acid that isnot native to the animal). The foreign nucleic acid may be present inall cells of the animal or in some but not all cells of the animal. Theforeign nucleic acid molecule is called a “transgene” and may containone or many genes, cDNA, etc. By inserting a transgene into a fertilizedoocyte or cells from the early embryo, the resulting transgenic animalmay be fully transgenic and able to transmit the foreign nucleic acidstably in its germline. Alternatively, a foreign nucleic acid may beintroduced by transferring, e.g., implanting, a recombinant cell ortissue containing the same into an animal to produce a partiallytransgenic animal. Alternatively, a transgenic animal may be produced bytransfer of a nucleus from a genetically modified somatic cell or bytransfer of a genetically modified pluripotential cell such as anembryonic stem cell or a primordial germ cell.

The term “intron” refers to a sequence of DNA found in the middle ofmany gene sequences in most eukaryotes. These intron sequences aretranscribed, but removed from within the pre-mRNA transcript before themRNA is translated into a protein. This process of intron removal occursby splicing together of the sequences (exons) on either side of theintron.

The term “operably-linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably-linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Similarly, when an intron isoperably-linked to a coding sequence, the intron is spliced out of themRNA to provide for expression of the coding sequence. In the context ofgene conversion, two nucleic acids sequences are operably linked if onesequence can “donate” sequence to the other by gene conversion. If twosequences are unlinked in that one can donate sequence to the other viagene conversion, the donating sequences may be upstream or downstream ofthe other, and the two sequences may be proximal to each other, i.e., inthat there are no other intervening genes. “Unlinked” means that theassociated genetic elements are not closely associated with one anotherand the function of one does not affect the other.

The terms “upstream” and “downstream” are used with reference to thedirection of transcription.

The term “pseudogene” is used to describe an untranscribed nucleic acidregion that contains an open reading frame that may or may not contain astart and/or a stop codon. An amino acid sequence may be “encoded” by apseudogene in the sense that the nucleotide sequence of the open readingframe can be translated in silico to produce an amino acid sequence.Pseudogenes do not contain promoter regions, recombination signalsequences or leader sequences.

A “transcribed gene” is a gene that is operably lined to a promoter andterminator, and has a coding sequence that is transcribed and translatedinto a protein product.

The term “homozygous” indicates that identical alleles reside at thesame loci on homologous chromosomes. In contrast, “heterozygous”indicates that different alleles reside at the same loci on homologouschromosomes. A transgenic animal may be homozygous or heterozygous for atransgene.

The term “native”, with reference to a gene or protein, indicates thatthe gene or protein is endogenous to a species, i.e., the gene ispresent at a particular locus in the genome of a non-modified organismof that species.

The term “construct” refers to a recombinant nucleic acid, generallyrecombinant DNA, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences. A constructmight be present in a vector or in a genome.

The term “recombinant” refers to a polynucleotide or polypeptide thatdoes not naturally occur in a host cell. A recombinant molecule maycontain two or more naturally-occurring sequences that are linkedtogether in a way that does not occur naturally. A recombinant cellcontains a recombinant polynucleotide or polypeptide. If a cell receivesa recombinant nucleic acid, the nucleic acid is “exogenous” to the cell.

The term “selectable marker” refers to a protein capable of expressionin a host that allows for ease of selection of those hosts containing anintroduced nucleic acid or vector. Examples of selectable markersinclude, but are not limited to, proteins that confer resistance toantimicrobial agents (e.g., hygromycin, bleomycin, or chloramphenicol),proteins that confer a metabolic advantage, such as a nutritionaladvantage on the host cell, as well as proteins that confer a functionalor phenotypic advantage (e.g., cell division) on a cell.

The term “expression”, as used herein, refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or ‘transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be incorporated into the genome of the cell (e.g.,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).

The term “replacing”, in the context of replacing one genetic locus withanother, refers to a single step protocol or multiple step protocol.

The term “coding sequence” refers to a nucleic acid sequence that oncetranscribed and translated produces a protein, for example, in vivo,when placed under the control of appropriate regulatory elements. Acoding sequence as used herein may have a continuous ORF or might havean ORF interrupted by the presence of introns or non-coding sequences.In this embodiment, the non-coding sequences are spliced out from thepre-mRNA to produce a mature mRNA. Pseudogenes may contain anuntranscribed coding sequence.

The term “in reverse orientation to” refers to coding sequences that areon different strands. For example, if a transcribed region is describedas being in reverse orientation to a pseudogene, then the amino acidsequence encoded by the transcribed region is encoded by the top orbottom strand and the amino acid sequence encoded by the pseudogene isencoded by the other strand relative to the transcribed region.

It is understood that the binding proteins produced by the presentmethod may have additional conservative amino acid substitutions whichhave substantially no effect on binding or other functions. Byconservative substitutions is intended combinations such as those fromthe following groups: gly, ala; val, ile, leu; asp, glu; asn, gln; ser,thr; lys, arg; and phe, tyr. Amino acids that are not present in thesame group are “substantially different” amino acids.

The term “specific binding” refers to the ability of a binding proteinto preferentially bind to a particular target that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable target in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

In certain embodiments, the affinity between a binding protein andtarget when they are specifically bound in an binding protein/targetcomplex is characterized by a K_(D) (dissociation constant) of less than10⁻⁶ M, less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than10⁻⁹ M, less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

As used herein the term “isolated,” when used in the context of anisolated protein, refers to protein that is at least 60% free, at least75% free, at least 90% free, at least 95% free, at least 98% free, andeven at least 99% free from other components with which the protein isassociated with prior to purification.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or ‘transformation”, or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be present in the cell transiently or may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon.

The term “plurality” refers to at least 2, at least 5, at least 10, atleast 20, at least 50, at least 100, at least 200, at least 500, atleast 1000, at least 2000, at least 5000, or at least 10,000 or at least50,000 or more. In certain cases, a plurality includes at least 10 to50. In other embodiments, a plurality may be at least 50 to 1,000.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of an antibody constitutes the basicstructural unit of an antibody. This form is a tetramer and consists oftwo identical pairs of antibody chains, each pair having one light andone heavy chain. In each pair, the light and heavy chain variableregions are together responsible for binding to an antigen, and theconstant regions are responsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv, andscFv fragments, chimeric antibodies, humanized antibodies, single-chainantibodies, and fusion proteins comprising an antigen-binding portion ofan antibody and a non-antibody protein. The antibodies may be detectablylabeled, e.g., with a radioisotope, an enzyme which generates adetectable product, a fluorescent protein, and the like. The antibodiesmay be further conjugated to other moieties, such as members of specificbinding pairs, e.g., biotin (member of biotin-avidin specific bindingpair), and the like. The antibodies may also be bound to a solidsupport, including, but not limited to, polystyrene plates or beads, andthe like. Also encompassed by the term are Fab′, Fv, F(ab′)₂, and orother antibody fragments that retain specific binding to antigen, andmonoclonal antibodies.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), andHunkapiller and Hood, Nature, 323, 15-16 (1986),).

An immunoglobulin light or heavy chain variable region consists of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been precisely defined (see, Lefrancet al, IMGT, the international ImMunoGeneTics information system.Nucleic Acids Res. 2009 vol. 37 (Database issue): D1006-12. Epub 2008Oct. 31; see worldwide website of imgt.org and referred to hereinafteras the “IMGT sytem”)). The numbering of all antibody amino acidsequences discussed herein conforms to the IMGT system. The sequences ofthe framework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs. The CDRs are primarilyresponsible for binding to an epitope of an antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, from antibodyvariable and constant region genes belonging to different species. Forexample, the variable segments of the genes from a chicken or rabbitmonoclonal antibody may be joined to human constant segments, such asgamma 1 and gamma 3. An example of a therapeutic chimeric antibody is ahybrid protein composed of the variable or antigen-binding domain from achicken or rabbit antibody and the constant or effector domain from ahuman antibody (e.g., the anti-Tac chimeric antibody made by the cellsof A.T.C.C. deposit Accession No. CRL 9688), although other mammalianspecies may be used.

As will be described in greater detail below, an antibody may be“classical antibody” or a “single chain antibody”.

For the purposes of this disclosure, a “classical antibody” is astereotypical “Y”-shaped molecule that consists of four polypeptidechains; two identical heavy chains and two identical light chainsconnected by disulfide bonds. Each chain is composed of an N-terminalvariable domain (V_(H) for the heavy chain and V_(L) for the lightchain) that is approximately 110 amino acids long and a C-terminalconstant domain (C_(H) for the heavy chain and C_(L) for the lightchain) that varies in type and length, depending on the type ofantibody. The heavy and light chains of a classical antibody are heldtogether by interactions between conserved cysteines (which occur in theheavy and light constant domains) and other charged amino acids.Sequence variability in a classical antibody is concentrated in theantigen binding site of the antibody, which are at the type of the armsof the Y. These regions are defined by the “complementarity-determiningregions” (“CDRs”) that are interspersed with regions that are moreconserved, termed “framework regions”. Each of the heavy and light chainvariable domains contains three CDRs (called CDR1, CDR2 and CDR3). In aclassical antibody, all six CDRs and both heavy and light variabledomains are required for antigen binding. Classical antibodies are madeby human, mice, rabbits, chicken and cattle, for example.

For the purposes of this disclosure, a “single chain antibody” is anantibody that contains an antigen binding site that is composed of asingle polypeptide chain. One example of a single chain antibody is asingle-chain variable fragment (scFv) antibody, which is a fusionprotein that contains the variable regions of the heavy (VH) and lightchains (VL) of a classical antibody connected by a short linker peptideof ten to about 25 amino acids. A single-chain antibody can also beobtained by immunization of a camelid (e.g., a camel, llama or alpaca)or a cartilaginous fish (e.g., a shark), which make antibodies that arecomposed of only heavy chains. A monomeric variable domain of a heavychain antibody binds antigen. The nucleotide sequence of a single chainantibody may be derived from a germline sequence or an mRNA sequence,for example. A classical antibody is not a single chain antibody becauseboth the heavy and light chains are required for antigen binding in aclassical antibody.

A “natural” antibody is an antibody in which the heavy and lightimmunoglobulins of the antibody have been naturally selected by theimmune system of a multi-cellular organism. Spleen, lymph nodes and bonemarrow are examples of tissues that produce natural antibodies in ananimal.

As used herein, the term “scaffold” refers to any monomeric protein(i.e., a protein that is composed of a single chain of amino acids thatis encoded by a single gene) that has a target binding domain and thatcan autonomously (i.e., without additional polypeptides) bind to atarget. A scaffold contains a “framework”, which is largely structural,and a “binding domain” which makes contact with the target and providesfor specific binding. The binding domain of a scaffold need not bedefined by one contiguous sequence of the scaffold. In certain cases, ascaffold may be part of larger binding protein, which, itself, may bepart of a multimeric binding protein that contains multiple scaffolds.Certain multimeric binding proteins may be bi-specific in that they canbind to two different epitopes. “Biparatopic” binding proteins can bindtwo distinct epitopes on the same target.

A scaffold may be derived from (i.e., have the same structure as but notnecessarily the same amino acid sequence as) a single chain antibody (asdefined above), or a scaffold may be not antibody-derived, in which acase it may have no sequence or structural relation to an antibodyvariable domain. Classical antibodies require both a heavy chain and alight chain for binding and, as such, do not contain a scaffold thatbinds to a target in the absence of additional polypeptides.

As used herein, the term “immunoglobulin heavy chain locus” is aposition of a genome that, in its wild-type form, encodes the heavychain of an antibody.

As used herein, the term “immunoglobulin light chain locus” is aposition of a genome that, in its wild-type form, encodes the lightchain of an antibody.

As used herein, the term “heavy chain constant region” is the constantregion of a heavy chain of an antibody.

As used herein, the term “not antibody derived” and grammaticalequivalents thereof in the context of a scaffold refer to a scaffoldthat has neither the characteristic structure of a variable domain of anantibody, nor a sequence of at least 100 contiguous amino acids that isat least 80% identical to an amino acid sequence in the variable domainof an antibody. The term “not antibody derived” is intended to excludesingle chain antibodies (i.e., the “only heavy chain” and scFvantibodies discussed above) as well as classical antibodies. Fibronectintype III domains (FN3's), Adnectins, DARPins, Affibodies, AvianPancreatic Peptides (APPs), Lipocalins, Atrimers, Kringle Domains,Phylomers, Centyrins,) etc. are examples of proteins that are notantibody derived. Other examples of scaffolds are described below.

As used herein, the term “gene conversion” refers to a well-knownmolecular phenomenon in which one allele of a sequence converts toanother base mismatch repair during recombination.

As used herein, the term “a light chain constant region but not a lightchain variable domain” and grammatical equivalents thereof refers to anantibody light chain that has been truncated to remove its variabledomain while retaining the constant domain. The constant domain of alight chain that lacks a variable domain is full length and can dimerizewith a heavy chain constant domain and produce a disulfide bondtherewith.

Further definitions may be elsewhere in this disclosure.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the present subject invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, and as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of cells and reference to “a candidate agent”includes reference to one or more candidate agents and equivalentsthereof known to those skilled in the art, and so forth. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely”, “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As noted above, a transgenic animal is provided. In certain embodiments,the animal may be any non-human animal that employs gene conversion fordeveloping their primary antigen repertoire and, as such, the animal maybe any of a variety of different animals. In one embodiment, the animalmay be a bird, e.g., a member of the order Galliformes such as a chickenor turkey, or a member of the order Anseriformes such as a duck orgoose, or a mammal, e.g., a lagamorph such as rabbit, or a farm animalsuch as a cow, sheep, pig or goat. In particular embodiments, thetransgenic animal may be a non-rodent (e.g., non-mouse or non-rat),non-primate transgenic animal.

Some of this disclosure may describe a transgenic chicken having a heavychain locus that contains a transcribed gene and pseudogenes. Since thenucleotide sequences of the immunoglobulin loci of many animals areknown, as are methods for modifying the genome of such animals, thegeneral concepts described below may be readily adapted to any suitableanimal, i.e., any animal that employs gene conversion for developingtheir primary antigen repertoire. The generation of antibody diversityby gene conversion between the variable region of a transcribedimmunoglobulin heavy or light chain gene and operably linked (upstream)pseudo-genes that contain different variable regions is described in avariety of publications such as, for example, Butler (Rev. Sci. Tech.1998 17: 43-70), Bucchini (Nature 1987 326: 409-11), Knight (Adv.Immunol. 1994 56: 179-218), Langman (Res. Immunol. 1993 144: 422-46),Masteller (Int. Rev. Immunol. 1997 15: 185-206), Reynaud (Cell 1989 59:171-83) and Ratcliffe (Dev. Comp. Immunol. 2006 30: 101-118).

As noted above, the genome of the subject transgenic animal may comprisea transcribed gene encoding a binding protein comprising, fromN-terminus to C-terminus: i. a scaffold comprising a first bindingdomain; and ii. a heavy chain constant region operably linked to thescaffold. The scaffold is characterized in that, unlike a classicalantibody, it is capable of specifically binding to a targetautonomously, i.e., in the absence of additional polypeptides. Thescaffold is not from a classical antibody. In certain embodiments, thescaffold may be the variable domain of a single chain antibody, or, inalternative embodiments, the scaffold is antibody-derived. Operablylinked to the transcribed gene is a plurality of untranscribedpseudogenes that donate, by gene conversion, nucleotide sequence to thepart of said transcribed gene that encodes the binding domain. Inparticular embodiments, the sequences may be arranged to parallel theendogenous heavy chain locus (shown in FIG. 1). As shown in FIG. 1, thesequences may be arranged in the same was as the endogenous heavy chainlocus in the animal, e.g., from 5′ to 3′, as follows: a) the pluralityof pseudogenes, b) a heavy chain promoter, c) an expressed scaffold thatis transcribed by the heavy chain promoter, d) an intron, and e) anative heavy chain constant region, although other arrangements arepossible.

In the transgenic animal, gene conversion between the bindingdomain-encoding part of the transcribed gene and the pseudogenes altersthe sequence of the scaffold by gene conversion, by as little as asingle nucleotide to multiple nucleotides distributed throughout theentire length of the binding domain, e.g., the entire length of thescaffold. Because: a) the arrangement of the sequences described hereinmimics the wild-type immunoglobulin heavy chain locus and b) the fusionprotein contains an endogenous constant domain, the scaffold is expectedto undergo selection and affinity maturation in a similar way toantibodies in an unmodified host animal. In other words, in a subjectanimal, the scaffold may be diversified in vivo through the geneconversion mechanism that is native to the animal, and upon immunologicchallenge, reactive lymphocytes may be selected via the animal's naturalcellular selection process. Since the C region is native to the animalsequence, a functional antigen receptor is formed in the milieu of otherproteins, and B lymphocyte development is normal. In certain cases, theanimal may be thought of as one in which the active V region andpseudo-V array of the heavy chain locus of the animal are replaced withalternative scaffold sequences, thereby allowing the animal to optimizethe interaction between the target and the binding domain of thealternative scaffold using the immune system of the animal. The fusionprotein produced by the transgenic animal is therefore encoded bywhatever sequence is donated from the pseudogenes into the transcribedgene. Clonal selection creates new sequences that are not encoded by thegermline and are unique to each clonal population of B lymphocytespresent within a single individual. Upon immunization, reactive clonescan be selected and enriched on the basis of their antigen receptor,which is a cell surface fusion protein encoded by the transcribed gene.After selection, sequences encoding an optimized scaffold can beisolated using conventional methods (e.g., using hybridoma technology orby PCR, etc).

The scaffold encoded by the subject gene may be a single chain antibody(as defined above). In other embodiments, the scaffold that is notantibody derived. Examples of scaffolds that are not antibody derivedinclude any non-antibody protein that is known to specifically bind to atarget, particularly a protein target. Suitable scaffolds are describedin Binz et al (Engineered proteins as specific binding reagents. CurrOpin Biotechnol. 2005 16:459-69), Binz et al (Engineering novel bindingproteins from nonimmunoglobulin domains. Nat. Biotechnol. 200523:1257-68), Forrer et al (Consensus design of repeat proteins.Chembiochem. 2004 5:183-9), Gronwall et al (Engineered affinityproteins—generation and applications. J. Biotechnol. 2009 140:254-69),Hosse et al (A new generation of protein display scaffolds for molecularrecognition. Protein Sci. 2006 15:14-27) and et al Skerra et al(Alternative non-antibody scaffolds for molecular recognition. Curr.Opin. Biotechnol. 2007 18:295-304), which are incorporated by referencefor disclosure of specific types of scaffolds, examples of wild typeproteins that are of a specific scaffold type and a description of thebinding domain of such scaffolds. Further details of the listedscaffolds, the positioning of the binding domain and which proteinscontain such binding domains, can be found in NCBI's conserved domaindatabase and NCBI's Genbank database, which database entries areincorporated by reference.

Scaffolds of particular interest include, but are not limited to:α-helical binding domains (e.g., those based on Z domain proteins suchas that from staphylococcal protein A; immunity proteins such as the E.coli colicin E7 and Im9 immunity proteins; Cytochrome b562 peptide; α2p8and repeat proteins such as ankyrin repeat proteins and leucine-richrepeat proteins), scaffolds with irregular secondary structures (e.g.,those based on insect defensin A; kunitz domain inhibitors such as BPTI,PSTI, APPI, LTDI, MTI II, ecotin, DX-88, LACI and HAE; PDZ domains suchas AF-6 and Omi; charybdotoxin; scorpion toxins; insect defensins; PHDfinger proteins such as CtBP2; TEM-1 and β-lactamase), and scaffoldswith β-sheet structures (e.g., those based on the 10th fibronectin typeIII domain (FNR); CTLA-4; T-cell receptors; knottins such as EETI-II,CBD, and Min-23; neocarzinostatin; carbohydrate binding module 4-2;tendamistat; lipocalins; and green fluorescent protein) as described inHosse, supra. Many other examples of suitable scaffolds are described inthe literature. The initial scaffold of the transcribed does not need tobind to a known target because gene conversion will modify the sequenceof the transcribed gene to produce a fusion protein that binds to thetarget. In particular cases, however, the initial scaffold may alreadybind to a known target. In these embodiments, the animal may in certaincases be employed to optimize binding to a target.

The number of pseudogenes upstream of the transcribed gene may varygreatly and in some embodiment may be in the range of 5 to 50, e.g., 10to 30 in number. The pseudogenes may be different to one another insequence, and may contain a number of point mutations that aredistributed throughout the pseudogene array. The pseudogenes generallycontain a nucleotide sequence that is at least 80% identical (e.g., atleast 90%, identical at least 95% identical at least 98% identical) toat least the part of the transcribed gene that encodes the bindingdomain of the scaffold. In some embodiments, the pseudogenes may containsequence that is related to only the binding domain encoding sequence ofthe scaffold. In other embodiments, the pseudogenes may contain sequencethat is related to more than the binding domain encoding sequence of thescaffold, e.g., 15 bases either side, 50 bases either side, 100 baseseither side or 200 bases either side, or more, up to entire length ofscaffold encoding sequence. The spacing between the pseudogenes mayvary. In certain embodiments, the spacing may be in the range of 50 to1,000 bases.

In particular embodiments, at least one (e.g., at least 2, at least 3,at least 5, at least 10 or more) of the plurality of pseudogenes may bein reverse orientation relative to the transcribed gene. In particularembodiments, the plurality of pseudogenes are not in alternatingorientations, and in certain cases may rather contain a series of atleast 5 or at least 10 adjacent pseudogene that are in oppositeorientation relative to the transcribed gene. In one embodiment, thepseudogene that is most distal from the transcribed variable region isin the same orientation as the transcribed gene, and the pseudogenebetween the most distal pseudogene and the transcribed gene are in thereverse orientation relative to the transcribed gene.

In addition to having an immunoglobulin heavy chain locus discussedabove, a transgenic animal in certain cases may also have a modifiedimmunoglobulin light chain locus. In one embodiment the immunoglobulinlight chain locus of the animal may be inactivated so that the animalproduces no light chain constant domain and, as such, the animalproduces only the fusion protein discussed above, i.e., without a lightchain constant domain-containing protein. In another embodiment, thegenome of the animal may contain an immunoglobulin light chain locusthat encodes only a light chain constant region, i.e., a light chainthat is not linked to a scaffold or variable domain. In theseembodiments, the fusion protein encoded by the heavy chain locus and thelight chain constant region encoded by the light chain locus, whenexpressed, link together via a disulfide bond. A resultant bindingprotein produced by this embodiment is illustrated in FIG. 2.

In a related embodiment, the animal may additionally comprise animmunoglobulin light chain locus comprising: a) a second transcribedgene encoding a second fusion protein comprising, from N-terminus toC-terminus: i. a second scaffold comprising a second binding domain; andii. a light chain constant region operably linked to the scaffold;wherein the scaffold is capable of specifically binding to a target inthe absence of additional polypeptides; and b) a plurality ofpseudogenes that are operably linked to the second transcribed gene andthat donate, by gene conversion, nucleotide sequence to the part of thesecond transcribed gene that encodes the second binding domain. In theseembodiments, the first and second binding domains may have differentbinding specificities. A resultant binding protein produced by thisembodiment is illustrated in FIG. 2. In particular cases, both the heavyand light chain immunoglobulin loci may be modified in a subject animal,and each arm of the resulting antibodies could have two independentbinding sites. For example, the variable domain of an endogenous locusmay be replaced with a scFv, and the pseudogene array would be comprisedof an array of different scFvs. This could be done at both the heavy andlight chain loci to achieve antibody-like molecules with dualspecificities.

In some instances one may modify the distance between the scaffold andthe constant region by using a linker sequence, and in some instancesthe scaffold may be so large that it is only possible to have one ofthem present on each arm of the antigen receptor. In this case, thelight chain locus may be modified to express only a truncated VL thatdoes not interfere with the bulky alternative scaffold that fused to theheavy chain C region.

In particular embodiments, part of the heavy chain locus, including theconstant region, part of an intron region and the 3′UTR of thetranscribed gene, may be endogenous to the animal and the remainder ofthe heavy chain locus, including the coding sequence of the transcribedgene, the remainder of the intron and the pseudogenes may be exogenousto the animal, i.e., made recombinantly and introduced into the animalproximal to the constant domain, part intron and 3′ UTR in such a waythat a transcribed gene is produced and the pseudogenes are capable ofdonating sequence to the transcribed gene by gene conversion. In certaincases the heavy chain locus of the animal may contain, in operablelinkage: an intron region, a constant domain-encoding region and a 3′untranslated region, where the intron region, the constantdomain-encoding region and the 3′ untranslated region are endogenous tothe genome of the transgenic animal, and a plurality of pseudogenes,where the plurality of pseudogenes are exogenous to the genome of thetransgenic animal. Alternatively, the constant domain encoding regioncould also be exogenous to the genome of the transgenic animal.

Along similar lines, the part of the light chain locus that includes theconstant domain-encoding region, part of an intron, and the 3′UTR of thetranscribed gene may be endogenous to the animal and the remainder ofthe light chain locus, including the coding sequence of the transcribedgene, the remainder of the intron and the pseudogenes may be exogenousto the animal, i.e., made recombinantly and introduced into the animalproximal to the constant domain, part intron and 3′ UTR in such a waythat a transcribed gene is produced and the pseudogenes are capable ofdonating sequence to the transcribed gene by gene conversion. In certaincases the light chain locus of the animal may contain, in operablelinkage: an intron region, a constant domain-encoding region and a 3′untranslated region; where the intron region, the constantdomain-encoding region and the 3′ untranslated region are endogenous tothe genome of the transgenic animal and a plurality of pseudogenes,where the plurality of pseudogenes are exogenous to the genome of thetransgenic animal.

A binding protein produced by a subject transgenic animal may contain anendogenous constant domain, allowing the binding protein to undergoclass switching and affinity maturation, which allows the animal toundergo normal immune system development, and mount normal immuneresponses. In specific embodiments transgenic chickens have threeendogenous constant regions in the heavy chain locus encoding IgM, IgYand IgA. During the early stages of B cell development, B cells expressIgM. As affinity maturation proceeds, class switching converts theconstant region into IgY or IgA. IgY provides humoral immunity to bothadults and neonatal chicks which receive about 200 mg of IgY via areserve deposited into egg yolk. IgA is found primarily in lymphoidtissues (eg. the spleen, Peyer's patches and Harderian glands) and inthe oviduct.

With the exception of a relatively small number of amino acids arisingas a result of mutations that occur independently of gene conversionduring affinity maturation (which occur in, e.g., less than 10%, lessthan 5%, less than 3%, or less than 1% of the amino acids), theresultant scaffolds produced by the transgenic animal may be differentto the initial scaffold by at least 1 amino acid, e.g., at least 5 aminoacids, at least 10 amino acids, at least 20 amino acids, or more, up toabout 50 amino acids. The resultant scaffold may bind to a target withat least 10×, e.g., at least 100×, at least 1000×, at least 10,000×, atleast 100,000× or at least 1,000,000× or more affinity than then theinitial scaffold.

The above-described transgenic animal may be made by replacing theendogenous variable regions in an endogenous immunoglobulin heavy chainlocus of an animal with a plurality of pseudogenes constructedrecombinantly. Methods for producing transgenic animals that use geneconversion to generate an antibody repertoire are known (see, e.g.,Sayegh, Vet. Immunol. Immunopathol. 1999 72:31-7 and Kamihira, Adv.Biochem. Eng. Biotechnol. 2004 91: 171-89 for birds, and Bosze,Transgenic Res. 2003 12:541-53 and Fan, Pathol. Int. 1999 49: 583-94 forrabbits and Salamone J. Biotechnol. 2006 124: 469-72 for cow), as is thestructure and/or sequence of the germline immunoglobulin heavy and lightchain loci of many of those species (e.g., Butler Rev Sci Tech 199817:43-70 and Ratcliffe Dev Comp Immunol 2006 30: 101-118), theabove-described animal may be made by routine methods given thisdisclosure. A strategy for making a subject animal is provided in FIG.3.

A method of making a transgenic animal is provided. In certainembodiments, the method comprises: replacing the variable regions in theendogenous immunoglobulin heavy chain locus of the animal with a) regionencoding a scaffold, as described above; and b) a plurality ofpseudogenes. Upon integration of the construct, the scaffold regionessentially becomes the transcribed variable region of theimmunoglobulin locus of the transgenic animal, and the pseudogenes alterthe sequence of the transcribed variable region by gene conversion. Geneconversion may result in the contribution of small (eg 1-10nucleotides), moderate (10-30 nucleotides), or large (>30 nucleotides)segments of DNA from one or more of the donor pseudogenes to thetranscribed scaffold. Gene conversion can transpire over manyiterations, so multiple pseudogenes may contribute sequence to thetranscribed gene. Since the process of gene conversion is highlyvariable in terms of which pseudogenes are selected, and the extent towhich each is utilized in a given lymphocyte, a large and diverseantibody repertoire will result in the transgenic animal. Similar changemay be made to the light chain locus, as described above.

As would be readily apparent, the method may include first deleting aregion containing the variable regions in the endogenous immunoglobulinheavy chain locus of the animal (including the transcribed variableregion and the pseudogene variable regions, and all sequences inbetween) to leave, e.g., a constant region sequence and part of theintron between the constant region sequence and the transcribed variableregion; and then adding the transcribed gene, the remainder of theintron, and the plurality of pseudogenes to the locus of the mammal.

In particular embodiments and as schematically illustrated in FIG. 3, atleast the variable region of the endogenous functional immunoglobulingene of the transgenic animal may be replaced by a nucleic acidconstruct containing a plurality of pseudogene variable regions and atranscribed gene, without replacing the endogenous pseudogene variableregions of the transgenic animal. As such, the resultant immunoglobulinlocus (which may be the heavy or light chain locus) may contain an arrayof endogenous pseudogenes in addition to an array of introducedpseudogenes upstream of a transcribed variable region.

Once made, the transgenic animal may be mated with other animals. Incertain cases, the animal may be mated with siblings to produce ananimal that is homozygous for the locus that produces no endogenousantibodies.

Once a subject transgenic animal is made, scaffolds that specificallybind to an antigen can be readily obtained by immunizing the animal withthe antigen. A variety of antigens can be used to immunize a transgenichost animal. Such antigens include, microorganism, e.g. viruses andunicellular organisms (such as bacteria and fungi), alive, attenuated ordead, fragments of the microorganisms, or antigenic molecules isolatedfrom the microorganisms. In certain embodiments, the animal may beimmunized with: GD2, EGF-R, CEA, CD52, CD20, Lym-1, CD6, complementactivating receptor (CAR), EGP40, VEGF, tumor-associated glycoproteinTAG-72 AFP (alpha-fetoprotein), BLyS (TNF and APOL—related ligand),CA125 (carcinoma antigen 125), CEA (carcinoembrionic antigen), CD2(T-cell surface antigen), CD3 (heteromultimer associated with the TCR),CD4, CD11a (integrin alpha-L), CD14 (monocyte differentiation antigen),CD20, CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD25(IL-2 receptor alpha chain), CD30 (cytokine receptor), CD33 (myeloidcell surface antigen), CD40 (tumor necrosis factor receptor), CD44v6(mediates adhesion of leukocytes), CD52 (CAMPATH-1), CD80 (costimulatorfor CD28 and CTLA-4), complement component C5, CTLA, EGFR, eotaxin(cytokine A11), HER2/neu, HERS, HLA-DR, HLA-DR10, HLA ClassII, IgE,GPiib/iiia (integrin), Integrin aVβ3, Integrins a4β1 and a4β7, Integrinβ2, IFN-gamma, IL-1β, IL-4, IL-5, IL-6R (IL6 receptor), IL-12, IL-15,KDR (VEGFR-2), lewisy, mesothelin, MUC1, MUC18, NCAM (neural celladhesion molecule), oncofetal fibronectin, PDGFβR (Beta platelet-derivedgrowth factor receptor), PMSA, renal carcinoma antigen G250, RSV,E-Selectin, TGFbeta1, TGFbeta2, TNFα, DR4, DR5, DR6, VAP-1 (vascularadhesion protein 1) or VEGF, or the like in order to produce atherapeutic scaffold. In a particular cases, the animal may be immunizedwith an antigen to which the scaffold already binds.

The antigens can be administered to a transgenic host animal in anyconvenient manner, with or without an adjuvant, and can be administeredin accordance with a predetermined schedule.

After immunization, serum or milk from the immunized transgenic animalscan be fractionated for the purification of pharmaceutical grade bindingproteins specific for the antigen. In the case of transgenic birds,antibodies can also be made by fractionating egg yolks. A concentrated,purified fraction may be obtained by chromatography (affinity, ionicexchange, gel filtration, etc.), selective precipitation with salts suchas ammonium sulfate, organic solvents such as ethanol, or polymers suchas polyethyleneglycol.

For making a monoclonal scaffold, antibody-producing cells, e.g., spleencells, may be isolated from the immunized transgenic animal and usedeither in cell fusion with transformed cell lines for the production ofhybridomas, or cDNAs encoding antibodies are cloned by standardmolecular biology techniques and expressed in transfected cells. Theprocedures for making monoclonal antibodies are well established in theart. See, e.g., European Patent Application 0 583 980 A1, U.S. Pat. No.4,977,081, WO 97/16537, and EP 0 491 057 B 1, the disclosures of whichare incorporated herein by reference. In vitro production of monoclonalantibodies from cloned cDNA molecules has been described byAndris-Widhopf et al., J Immunol Methods 242:159 (2000), and by Burton,Immunotechnology 1:87 (1995), the disclosures of which are incorporatedherein by reference.

As such, in addition to the transgenic animal, a method comprisingimmunizing the transgenic animal with an antigen and obtaining from thetransgenic animal a scaffold that specifically binds to the antigen isalso provided. The method may include making hybridomas using cells ofthe transgenic animal; and screening the hybridomas to identify ahybridoma that produces a scaffold that specifically binds to theantigen.

Compositions comprising a fusion protein are also provided. In theseembodiments, the fusion protein may comprise, from N-terminus toC-terminus: i. a scaffold comprising a first binding domain, asdescribed above; and ii. a heavy chain constant region operably linkedto the scaffold, as described above. As noted above, the scaffold is notfrom a classical antibody and the scaffold specifically binds to aselected target in the absence of additional polypeptides. The fusionprotein may exist on its own, or complexed with one or more otherproteins. In particular embodiments, the fusion protein may exist in acomplex that may comprise a light chain protein that comprises a lightchain constant region but not a light chain variable domain, wherein thelight chain constant region and the heavy chain constant region of thefusion protein are linked by a disulfide bond. In additionalembodiments, the fusion protein may exist in a complex that comprises alight chain protein that comprises: i. a scaffold comprising a secondbinding domain; and ii. a light chain constant region operably linked tothe scaffold. In these embodiments, the scaffold is connected to thelight chain constant region specifically binds to a selected target inthe absence of additional polypeptides, and the light chain constantregion and the heavy chain constant region are linked by a disulfidebond. As would be readily apparent, the binding specificities of thescaffold attached to the heavy chain constant region and the scaffoldattached to the light chain constant region may be different and, assuch, this protein may be bispecific in that it binds to two distinctmolecular targets, or “biparatopic” (i.e. binding two distinct epitopeson the same molecular target).

Aspects of the present teachings can be further understood in light ofthe following example, which should not be construed as limiting thescope of the present teachings in any way.

EXAMPLE

Alternative scaffold heavy chain and truncated light chain expressionconstructs (designed to produce a protein illustrated at the top of FIG.2) were co-transfected into HEK 293 cells, and secreted product wasrecovered and run on SDS-PAGE under non-reducing conditions. The gel waselectroblotted onto PVDF membrane and probed with anti-chicken IgYantibody. This heavy chain contained autonomous “camelized” human VHgene linked to the C regions of IgY. The light chain had a leaderpeptide linked directly to CL (100% truncation of VL). The expressedproduct migrated at the expected molecular weight (˜160 kD) for dimericheavy chain paired with light chain. The results (shown in FIG. 4)confirm that truncated light chain can support the proper processing andsecretion of novel scaffolds when they are genetically fused to theappropriate heavy chain constant regions.

1-22. (canceled)
 23. A transgenic chicken comprising a genome comprisingan immunoglobulin heavy chain locus comprising: (a) a transcribed geneencoding a fusion protein comprising, from N-terminus to C-terminus: abinding domain and, operably linked to said binding domain, at leastpart of a heavy chain constant region that is native to said transgenicchicken; and (b) a plurality of pseudogenes that are operably linked tosaid transcribed gene and that donate, by gene conversion, nucleotidesequence to the part of said transcribed gene that encodes said bindingdomain, wherein the pseudogenes are upstream or downstream of thetranscribed gene and contain a nucleotide sequence that is at least 80%identical to at least part of the transcribed gene; wherein the genomeof the chicken further comprises an immunoglobulin light chain locusthat encodes a light chain constant region but not a light chainvariable domain, and wherein the transgenic chicken produces adiversified population of fusion proteins whose binding specificity issolely determined by a diversified binding domain of (a).
 24. Thetransgenic chicken of claim 23, wherein the binding domain is anantibody binding domain.
 25. The transgenic chicken of claim 24, whereinthe antibody binding domain is a single chain antibody.
 26. Thetransgenic chicken of claim 23, wherein the binding domain is not anantibody binding domain.
 27. The transgenic chicken of claim 26, whereinthe binding domain comprises a binding domain of fibronectin type III,an adnectin binding domain, a DARPin binding domain, an affibody bindingdomain, an avian pancreatic peptide binding domain, a lipocalin bindingdomain, an atrimer binding domain, a kringle binding domain, a phylomerbinding domain, a centyrin binding domain, or a knottin binding domain.28. The transgenic chicken of claim 23, wherein the nucleotide sequencesof said pseudogenes are at least 90% identical to the nucleotidesequence of said part of said transcribed gene.
 29. The transgenicchicken of claim 23, wherein said immunoglobulin heavy chain locuscomprises at least 10 of said pseudogenes.
 30. The transgenic chicken ofclaim 23, wherein said transgenic animal is made by replacing theendogenous variable region in an endogenous immunoglobulin heavy chainlocus of the transgenic chicken with a nucleic acid construct comprisingsaid plurality of pseudogenes and encoding said binding domain, withoutreplacing all of the constant region of said endogenous immunoglobulinheavy chain locus.
 31. The transgenic chicken of claim 23, wherein atleast one of said plurality of pseudogenes is in reverse orientationrelative to said transcribed gene.
 32. A method comprising: immunizing atransgenic chicken of claim 23 with an antigen; and obtaining from saidtransgenic chicken a diversified population of fusion proteins that areencoded by said immunoglobulin heavy chain locus and that comprise atleast some fusion proteins that specifically binds to said antigen. 33.The method of claim 32, further comprising: obtaining a fusion proteinthat binds to said antigen from said chicken.
 34. The method of claim33, wherein the method comprises: making hybridomas using cells of saidtransgenic chicken; and screening said hybridomas to identify ahybridoma that produces a fusion protein that specifically binds to saidantigen.
 35. The method of claim 33, further comprising: humanizing saidfusion protein.
 36. The method of claim 32, further comprising using PCRto amplify nucleic acid that encodes at least the binding domain of saidfusion protein from a lymphocyte of said transgenic chicken, andexpressing a recombinant protein that binds to the antigen using saidamplified nucleic acid.
 37. An isolated cell of a transgenic chicken ofclaim 23, wherein the cell produces a fusion protein whose bindingspecificity is determined by its binding domain.
 38. A hybridoma made byfusing an isolated cell of claim 37 and another cell.